Process for preparing pectin from plant tissues

ABSTRACT

A process for preparing pectin, which is useful for foods and medicines or the like, by subjecting a plant tissue containing pectic substances to the action of a microorganism which belongs to the genus Endomyces, Endomycopsis, Saccharomyces, Shizosaccharomyces, Pichia, Hansenula, Debaryomyces, Hanseniaspora, Torulopsis, Candida or Kluyveromyces and possesses an activity liberating pectin from a plant tissue; or of a culture broth or processed material thereof to liberate pectin from said plant tissue; and recovering pectin.

The present invention relates to a useful method for the isolation ofpectin from plant tissues with the use of particular yeasts.

Pectin is a useful polysaccaride utilized as a raw material for foods,medicines or cosmetics, which is contained in higher plants in largeamounts. Hitherto, the production of pectin has been carried out byheat-extracting a plant tissue containing pectic substances in thepresence of a chelating agent, an acid or the like. However, suchmethods cause difficulty in the isolation of pectin and problems in thetreatment of residues.

The present inventors have repeatedly studied methods for producingpectin under mild conditions, instead of the known chemical methodsmentioned above, and have previously found that Trichosporonpenicillatum SNO-3, belonging to the genus Trichosporon, produces anenzyme which is able to liberate pectin from various plant tissues,without rupturing the tissues (Japanese Unexamined Patent PublicationNo. 70495/1979). In that application, a process for preparing pectin bysubjecting a plant tissue containing pectin as a component to theactions of a microorganism which belongs to the genus of Trichosporonand possesses an activity of liberating pectin from a plant tissue, aculture broth of said microorganism and its extract is claimed. As theresult of a further development for the above studies, it was found thatvarious yeasts possess an activity of liberating pectin from planttissues containing pectic substances, which is the basis of the presentinvention.

Thus, according to the present invention, there is provided a processfor preparing pectin, which is characterized by subjecting a planttissue containing pectic substances to the action of a microorganismwhich belongs to the genus Endomyces, Endomycopsis, Saccharomyces,Schizosaccharomyces, Pichia, Hansenula, Debaryomyces, Hanseniaspora,Torulopsis, Candida or Kluyveromyces and possesses an activityliberating pectin from a plant tissue; or of a culture broth orprocessed material thereof to liberate pectin from said plant tissue;and recovering the pectin.

The microorganisms used in the invention are sporogeneous or sporogenousyeasts. Examples thereof are as follows.

The microorganisms belonging to Endomyces are Endomyces geotrichum andEndomyces lindneri;

those belonging to Endomycopsis are Endomycopsis capsularis andEndomycopsis vernalis;

those belonging to Saccharomyces are Saccharomyces uvarum, Saccharomycesbailii, Saccharomyces delbrueckii and Saccharomyces fermentati;

those belonging to Schizosaccharomyces is Schizosaccharomycesoctosporus;

those belonging to Pichia are Pichia orientalis, Pichia polymorpha andPichia farinosa;

those belonging to Hansenula are Hansenula saturnus and Hansenulaminuta;

those belonging to Debaryomyces are Debaryomyces hansenii andDebaryomyces castellii;

those belonging to Hanseniaspora are Hanseniaspora valbyensis andHanseniaspora uvarum;

those belonging to Torulopsis are Torulopsis sphaerica and Torulopsispinus;

those belonging to Candida are Candida krusei, Candida glaebosa andCandida macedoniensis;

those belonging to Kluyveromyces are Kluyveromyces fragilis,Kluyveromyces lactis, Kluyveromyces marxianus and Kluyveromycesdrosophilarum; and strains similar to these strains and mutants thereof.The term "strains similar to these strains" mentioned above means thosewhich possess a pectin-liberating activity and have mycologicalproperties similar to those of these strains. The term "mutants thereof"also includes any of the mutants of the specifically exemplified strainsand strains similar thereto which are artifically induced by chemical orphysical means or are spontaneously induced. Artificial means which maybe used for the mutation are those known in the art.

The present invention also depends upon the discovery that yeastmicroorganisms contain an enzyme which is capable of liberating pectin.

Any strains to be included in the above-mentioned genera, insofar asthey contain such an enzyme, are within the scope of the presentinvention.

For the isolation of pectin from a plant tissue according to the methodof the present invention, the above microorganisms may be directlyinoculated into a plant tissue to be treated and cultured under static,agitating or shaking conditions in accordance with conventional methods;or a culture broth which is obtained by cultivating the abovemicroorganism or processed material thereof may be contacted with aplant tissue. Media to be used for the cultivation of the microorganism,i.e., the seed cultivation is not particularly limited and may be anyone which contains the various nutrients normally used in theconventional cultivation of yeasts. The usual media may suitably containpeptone, casein hydrolysate, yeast extract and glucose and, ifcircumstances require, inorganic salts, such as phosphates, magnesiumsalts or potassium salts. Certain kinds of plant tissues may also beused as the seed cultivation medium, without the addition of anynutrients but after heat sterilization.

The peel or segment cover of citrus fruits is an especially advantageousmedium for this purpose.

The cultivation conditions for the microorganism in the above-mentionedmedium, are properly determined to maximize the quantity of productionof the intended enzyme, usual conditions being 20°-37° C. for 10-50hours. The cultivation may be with shaking, static or aeration-stirringor in a solid state. Under such cultivation conditions, mycelium isgrown and an enzyme is liberated from the mycelium. The cultivationbroth may be utilized as such or as processed material, as a concentrateor as a solution of purified enzyme. The concentrate may be obtained bysubjecting the cultivation broth, which is, if necessary, filtered, to atreatment under mild conditions, e.g., evaporating the broth at a lowtemperature and low pressure or concentrating the broth by the use of anultra-filtration membrane. The solution of purified enzyme may also beobtained from the broth, e.g., which is filtrated, concentrated and thensubjected to repeated fractional precipitation by the addition ofammonium sulphate; alternatively, it may be filtered and subjected toCM."Sephadex" column chromatography and then to "Sephadex" G-75 gelfiltration.

The mechanism involved in the method of the present invention isconsidered to be one in which an enzyme produced from the yeastpenetrates into a plant tissue, acts on pectic substances, especiallywater-insoluble protopectin, and produces water-soluble pectin, pectinthereby being liberated from the plant tissue.

Pectin liberated from plant tissue by the method of the presentinvention may be isolated in accordance with conventional methods. Forexample, the solution, treated as mentioned above, is filtered and thefiltrate is mixed with three times its volume of a water-miscibleorganic solvent, e.g., ethanol, to precipitate out pectin. The collectedpectin is washed with a solvent similar to the above organic solvent,e.g., ethanol, and dried to give pectin of high purity.

Pectin thus obtained has a molecular weight of more then 100,000,irrespective of the kind of yeast used. The pectins are different fromthose obtained by the known chemical methods. They possess a narrowmolecular weight distribution and are similar to natural pectin.Furthermore, the pectin is suitable for use as a food or medicine, sinceit does not contain chemical substances. In addition, the properties ofpectins obtained by the invention vary more or less, according to thenature of the raw materials.

The present invention relates to the method of obtaining a high purityof pectin in a simple way and in a high yield from plant tissues, by theutilization of yeast microorganisms, as described above, and also to amethod which can be utilized for the removal of pectin from planttissues or for the production of an extract of plant containing pectin.

Furthermore, the plant tissues used in the treatment of the presentinvention are preferably those available at a low price and with as higha pectin content as possible, although they are not particularly limitedthereto. Examples are citrus fruits, such as Citrus unshiu, Citrusnatsu-daidai, lemon, grapefruit, navel orange or the like. Any of thepeels and the segment covers of these citrus fruits may be used as theraw material. It is also possible to use the residue which is obtainedby pressing citrus fruits to obtain fruit juice, and which is,therefore, cheap and serves as a utilization of waste material.

The invention is illustrated by the following examples:

EXAMPLE 1

500 g of finely cut fresh peel of Citrus unshiu were suspended in 1 l ofsterilized water in a sterilized flask, and 50 ml of each of thecultivation broths of the various strains of Table 1 were added to thesuspension. The resultant was incubated at 30° C. for 24 hours withoccasional stirring, and filtered through a gauze. To the supernatantwas added three times its volume of ethanol to precipitate pectin,followed by drying it in vacuo. The results obtained are shown in Table1.

Each of the seed cultivations was conducted with the use of a medium (pH5.0) containing 0.5% of peptone, 3% of glucose, 0.2% of CaCl₂, 0.1% ofKH₂ PO₄, 0.05% of MgSO₄.7H₂ O at 30° C. for 24 hours, with shaking.

EXAMPLE 2

Each of various strains used in Example 1 was cultivated under the samecondition as in Example 1, except for prolonging it to 36 hours. 300 gof segment cover of Citrus natsudaidai were added to 300 ml of each ofthe cultivation filtrates and incubated, with shaking, at 30° C. for 6hours. The reaction solution was filtered and three times its volume ofethanol were added to the filtrate to precipitate the pectin obtained.It was collected and dried in vacuo. The yields are shown in Table 2.

EXAMPLE 3

Kluyveromyces marxianus (IFO 0277) was cultivated under the sameconditions as in Example 1. 100 g of each of various plant tissues wereadded to 100 ml of the cultivation filtrate broth and incubated for 3hours, followed by the same treatments as in Example 1, to give pectin.The yields are as shown in Table 3.

EXAMPLE 4

In accordance with the method of Example 1, Endomycopsis capsularis (IFO0672) was allowed to act on the peel of Citrus unshiu. The treatedsolution was filtered and to the filtrate was added three times itsvolume of ethanol, to precipitate pectin. It was collected, washed wellwith ethanol and dried to give pectin having the properties shown inTable 4.

                  TABLE 1                                                         ______________________________________                                        Yields of pectin obtained from the peel of                                    Citrus unshiu                                                                 Strain                   Yield of pectin                                      ______________________________________                                        Candida krusei IFO 0013  4.0     (g)                                          Candida glaebosa IFO 1353                                                                              0.7                                                  Candida macedoniensis AKU 4587                                                                         0.7                                                  Debaryomyces hansenii IFO 0794                                                                         4.2                                                  Debaryomyces castellii IFO 1359                                                                        0.5                                                  Endomyces geotrichum IFO 9541                                                                          6.8                                                  Endomyces lindneri AKU 4206                                                                            0.8                                                  Hanseniaspora valbyensis IFO 0115                                                                      3.0                                                  Hanseniaspora uvarum IFO 1413                                                                          0.5                                                  Hansenula saturnus IFO 0117                                                                            4.5                                                  Hansenula minuta IFO 0975                                                                              4.0                                                  Kluyveromyces fragilis IFO 0288                                                                        6.3                                                  Kluyveromyces lactis IFO 1090                                                                          4.0                                                  Kluyveromyces marxianus IFO 0277                                                                       9.0                                                  Kluyveromyces drosophilarum IFO 1012                                                                   4.0                                                  Pichia orientalis IFO 1279                                                                             3.2                                                  Pichia polymorpha AKU 4250                                                                             0.6                                                  Pichia farinosa AKU 4251 0.6                                                  Saccharomyces uvarum IFO 0565                                                                          3.0                                                  Saccharomyces bailii IFO 1047                                                                          3.0                                                  Saccharomyces delbrueckii IFO 0285                                                                     4.2                                                  Saccharomyces fermentati IFO 0422                                                                      4.8                                                  Schizosaccharomyces octosporus IFO 0353                                                                4.1                                                  Torulopsis sphaerica IFO 0648                                                                          5.3                                                  Torulopsis pinus IFO 0741                                                                              0.8     (g)                                          Enxomycopsis capsularis IFO 0672                                                                       7.5                                                  Endomycopsis vernalis AKU 4210                                                                         0.5                                                  ______________________________________                                    

                  TABLE 2                                                         ______________________________________                                        Yields of pectin obtained from the segment cover of                           citrus natsudaidai                                                            Strain                   Yield of pectin                                      ______________________________________                                        Candida krusei IFO 0013  2.5     (g)                                          Debaryomyces hansenii IFO 0794                                                                         2.3                                                  Endomyces geotrichum IFO 9541                                                                          3.7                                                  Hanseniaspora valbyensis IFO 0115                                                                      1.6                                                  Hansenula saturnus IFO 0117                                                                            2.2                                                  Hansenula minuta IFO 0975                                                                              1.9                                                  Kluyveromyces fragilis IFO 0288                                                                        3.5                                                  Kluyveromyces lactis IFO 1090                                                                          2.8                                                  Kluyveromyces marxianus IFO 0277                                                                       5.0                                                  Kluyveromyces drosophilarum IFO 1012                                                                   2.4                                                  Pichia orientalis IFO 1279                                                                             1.5                                                  Saccharomyces uvarum IFO 0565                                                                          1.3                                                  Saccharomyces bailii IFO 1047                                                                          1.5                                                  Saccharomyces delbrueckii IFO 0285                                                                     2.9                                                  Saccharomyces fermentati IFO 0422                                                                      3.0                                                  Schizosaccharomyces octosporus IFO 0353                                                                2.2                                                  Torulopsis sphaerica IFO 0648                                                                          2.3                                                  Endomycopsis capsularis IFO 0672                                                                       4.0                                                  ______________________________________                                    

                  TABLE 3                                                         ______________________________________                                        Yields of pectin obtained from the peel or                                    segment cover of citrus fruits by the use                                     of Kluyveromyces marxianus IFO 0277                                           Fruit                Yield of pectin                                          ______________________________________                                        Navel orange                                                                  Peel                 1.9     (g)                                              Segment cover        1.6                                                      Grapefruit                                                                    Peel                 1.9                                                      Segment cover        1.3                                                      Citrus unshiu                                                                 Peel                 1.8                                                      Segment cover        1.2                                                      Citrus natsudaidai                                                            Peel                 1.9                                                      Segment cover        1.7                                                      Lemon                                                                         Peel                 1.0                                                      Segment cover        1.1                                                      ______________________________________                                    

                  TABLE 4                                                         ______________________________________                                        Properties of pectin obtained from the peel                                   of Citrus unshiu by the use of Endomycopsis                                   capsularis IFO 0672                                                           ______________________________________                                        Moisture            8.6%                                                      Ash*               ≦0.01%                                              Methoxylated carboxyl group                                                                      85.4%                                                      Galacturonic acid  75.0%                                                      Neutral sugar      25.0%                                                      Relative viscosity 1.52                                                       (0.1% solution)                                                               pH (0.5% solution) 3.3                                                        Molecular weight** 105,000                                                    Found values by    C: 40.25, H: 5.78                                          elementary analysis                                                                              and N: 0.60%                                               ______________________________________                                         *Measured on a sample without ion exchange treatment.                         **Determined by the method of Smit and Bryant [J. Food Science, 32, 197,      (1967)].                                                                 

We claim:
 1. A process for preparing pectin comprising subjecting aplant tissue containing pectin substances to the action of amicroorganism selected from Kluyveromyces fragilis IFO 0288 orKluyveromyces marxianus IFO 0277 which possesses an activity ofliberating pectin from a plant tissue or to the action of a culturebroth or processed material of said microorganism which possesses anactivity of liberating pectin from plant tissue to liberate pectin fromsaid plant tissues, and recovering the pectin.
 2. A process as describedin claim 1, wherein the peel or segment cover of citrus fruits is usedas the plant tissue containing pectic substances.
 3. A process asdescribed in claim 2, wherein the citrus fruit is Citrus unshiu, Citrusnatsudaidai, grapefruit, orange or lemon.